The menace of bioterrorism and the emergence of antibiotic resistance in an increasing number of pathogenic bacterial highlight the need for the development of novel classes of anti-infective agents. New antibiotic development needs to focus on novel targets and will require a basic understanding of their cellular function if we are to prevent and combat the insidious threat of bioterrorism. Promisingly, bacteria possess a unique target (the SmpB-tmRNA mediated ribosome rescue, protein tagging and directed degradation system) that we have recently shown to be essential for the pathogenesis and survival of Yersinia in the micro-niches of its host. The objective of our research project is to understand the SmpB-tmRNA system, to probe its mechanism of ribosome rescue, protein tagging and nonstop mRNA decay, and to determine how bacterial proteins tagged by the tmRNA system are recognized and degraded by bacterial AAA+ proteases. Principally, through these studies we wish to investigate the structure, function, and substrate binding specificity of Lon, a major protease that degrades tmRNA-tagged proteins. A detailed knowledge of Lon- substrate range and specificity will provide new insight into how this protease contributes to the fitness, survival and virulence of two category-A pathogens,/, pestis and F. tularensis. The Lon ATP-dependent protease could also serve as unique model for understanding molecular mechanisms that have been conserved from bacteria to humans. Our investigations will be conducted in close collaboration with the laboratories of Dr. Jim Bliska (Project 1) and Dr. Martha Furie (Project 2). To assist us in these studies, we will develop high-throughput in vitro and in vivo assays that will enable us to scrutinize the mechanistic details of the SmpB-tmRNA mediated ribosome rescue process. These assays will also be used to screen chemical libraries for small molecules that specifically inhibit SmpB-tmRNA interaction and/or the trans- translation process. A deeper understanding of this unique bacterial surveillance process will not only reveal new functional aspects of the SmpB-tmRNA system, protein synthesis and selective proteolysis but likely yield new insights into how antibiotics function and how novel anti-infectives could be discovered. RELEVANCE (See instructions): Plague and tularemia are highly virulent diseases that are considered major threats as biological weapons. Current strategies to prevent them, such as the use of vaccines or antibiotics, are non-effective or could be subverted by intentional genetic manipulation of the pathogen. The studies proposed here will foster the development of new strategies to prevent or treat plague and tularemia in the human population.